Abstract: Approximately 15-20% of breast cancers have a triple negative phenotype (negative for ER and PR expression and lack ErbB2 overexpression) that correlates with aggressive cancer and limited treatment options. We have developed an in vivo screen for defining the role of specific kinases in tumorigenesis and metastasis of breast cancer cells. Triple negative and triple positive breast cancer lines were used for shRNA-mediated knock down of specific kinases. The genetically altered lines (expressing luciferase in addition to specific shRNA gene knockdown) are injected into the mammary fat pad of female SCID mice. Tumor growth and vascularization is monitored longitudinally over an 8 week period using a micro-probe ultrasound system. Metastasis is monitored by bioluminescence imaging. The assay provides an in vivo screen for analysis of proteins that control the growth, vascularization and metastasis of breast tumors. MAP3Ks are the first tier of kinases regulating the MAP kinase signaling pathways that lead to the activation of the MAPKs ERKl/2, p38, JNK and ERKS. MAPSKs control expression of genes important for regulating the cell cycle, cytokine and protease expression and apoptosis. In a screen of 9 MAP3Ks, MEKK2 was identified as a key regulator of metastasis using MDA-MB-231 (triple negative basal) and BT474 (triple positive luminal) breast adenocarcinoma cells in the in vivo tumorigenesis assay. MEKK2 is a MAP3K that regulates the activation of the JNK and ERK5 pathways via activation of MKK7 and MEKS. We have shown that MEKK2 expression is required for EGFR (ErbBI) and ErbB2/Neu activation of ERKS in MDA-MB-231 and BT474 cells, respectively. Our hypothesis is that the MAP3K MEKK2 functions as a critical signaling node within the cell signaling network stimulating tumor growth and metastasis in response to ErbB and possibly other tyrosine kinases. The goal of this proposal is to genetically define the role of MEKK2 in triple negative breast cancer tumor growth and metastasis and to develop a MEKK2 small molecule inhibitor. Specific aim 1 involves defining the role of MEKK2-MEK5-ERK5 signaling in tumorigenesis and metastasis of triple negative breast cancer cells using the in vivo xenograft assay. In specific aim 2, we propose to elucidate the mechanism by which MEKK2 gets activated by ErbB1/2 using MEKK2 mutants in biochemical and cell-based activation assays. In specific aim 3, a small molecule biochemical screen will be developed to identify compounds that specifically inhibit MEKK2 kinase activity. MEKK2 inhibitors will be tested for MAPK pathway specificity in cell-based assays and profiled for specificity against the kinome. In future studies, these MEKK2 inhibitors will be tested for anti-tumor efficacy in genetically engineered mouse models (GEMMs) of breast cancer.